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In my PhD thesis, I used two‐photon imaging to investigate neuronal circuits and glia cells in the spinal cord of living mice. To achieve this, a major effort first was to establish a mouse spinal cord preparation suitable for stable and long‐lasting imaging experiments. Without adequate stabilisation, the spinal cord was prone to large‐scale movement artefacts clearly hampering high‐resolution imaging in vivo. To overcome these limitations, I employed strategies to optimise the animals posture, namely rigid clamping of the vertebral column to isolate the spinal cord from breathing‐related movements. In addition, I developed strategies to dampen tissue movements remaining after posture optimisation. These improvements made it possible to image the structural plasticity of genetically labelled microglia cells with subcellular resolution for many hours in anesthetized mice. In a paradigm of focal spinal cord injury, microglia became rapidly activated and displayed high levels of filopodial motility clearly directed towards the injury site. In addition, I adapted Ca2+ indicator loading techniques to stain neuronal networks in the mouse superficial dorsal horn to visualize activity patterns of painprocessing neurons. Despite the heavily myelinated surrounding tissue, neuronal populations within the first two laminae could be visualized after Ca2+ indicator loading. The preparation was sufficiently stable to for the first time resolve fast, individual Ca2+ transients in the spinal cord of living rodents. In agreement with the role of dorsal horn circuits in nociceptive processing I found low rates of spontaneous activity but could reliably evoke increased activity levels by electrical stimulation of primary afferent fibres in situ. Specifically, also natural sensory stimulation applied to the paw elicited Ca2+ transients in a subset of dorsal horn neurons. In a parallel project, I collaborated with Klas Kullander’s group to investigate activity patterns of identified Ren